The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering B.t. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner and Kim, 1988). Thus, isolated B.t. endotoxin genes are becoming commercially valuable.
Until the last ten years, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of B. thuringiensis var. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thudngiensis var. kurstaki HD-1 produces a crystal called a delta endotoxin which is toxic to the larvae of a number of lepidopteran insects.
In recent years, however, investigators have discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of B.t., namely B.t. var. israelends and B.t. var. tenebdonis (a.k.a. M-7, a.k.a.B.t. var. san diego), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, 1989). See also Couch, 1980 and Beegle, 1978. Krieget al, 1983, describe Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineata, and the beetle Agelastica alni.
Recently, new subspecies of B.t. have been identified, and genes responsible for active .delta.-endotoxin proteins have been isolated (Hofte and WhiteIcy, 1989). Hofte and Whiteley classified B.t. crystal protein genes into 4 major classes. The classes were CryI (Lepidoptera-specific), CrylI (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). Prefontaine et al., 1987, describe probes useful in classifying lepidopteran-active genes. The discovery of strains specifically toxic to other pests has been reported (Feitelson et al., 1992).
B.t. crystalline toxins are generally recognized as being protoms, requiring either particular physicochemical conditions (i.e., pH, redox, ionic strength), or the action of certain proteases, or both, to generate an active toxin (Hofte and WhiteIcy, 1989). In most cases, the insect supplies conditions for activation of the toxin; however, cases have been documented where pre-solubilization or pre-proteolysis have been necessary for optimum activity (Jacquet et al., 1987) or detection of activity (Hofte et al., 1992).
The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described in the published literature (Schnepf and Whiteley, 1981). U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose the expression of B.t. crystal proteins in E. coli. U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose B. thuringiensis var. tenebrionis (a.k.a.B.t. san diego, a.k.a. M-7) which can be used to control coleopteran pests in various environments. U.S. Pat. No. 4,918,006 discloses Bacillus thuringiensis var. israelensis toxins which are active against dipteran pests. This patent reports that a protein of about 27 kD, and fragments thereof, are responsible for the dipteran activity. U.S. Pat. No. 4,849,217 discloses B.t. isolates which have activity against the alfalfa weevil. U.S. Pat. No. 5,151,363 and U.S. Pat. No. 4,948,734 disclose certain isolates of B.t. which have activity against nematodes. As a result of extensive research and investment of resources, other patents have issued for new B.t. isolates and new uses of B.t. isolates. However, the discovery of new B.t. isolates and new uses of known B.t. isolates remains an empirical, unpredictable art.
Cockroaches such as the German cockroach (Blatella germanica), Oriental cockroach (Blatta orientalis), American cockroach (Periplaneta americana) and Brown cockroach (Periplaneta americana) are some of the most important insect pests to infest homes and commercial structures. These pests have omnivorous feeding habits resulting in the destruction of food, leather and fabrics. Cockroaches have also been implicated in the transmission of the disease-causing organisms Salmonella and Toxoplasma. Additionally, cockroaches contain altergins, to which approximately 7.5 percent of the human population are sensitive.
Although B.t. toxicity to cockroaches has been reported (Singh and Gill, 1985), this toxicity could only be achieved by directly injecting solubilized B.t. crystals into the foregut or hemocoel of the cockroaches. Attempts to feed or force-feed B.t. preparations to the cockroaches were unsuccessful.
Cockroaches are frequently controlled with insecticidal baits. Because these baits are used in homes and restaurants, cockroach insecticides must be safe to humans. The heavy use of synthetic insecticides has resulted in the selection of resistant cockroach populations. New insecticides, safe for use around humans, are necessary to control these resistant populations.